Exercise hyperemia is a biomedically significant phenomenon because skeletal muscle blood flow is a key determinant of exercise capacity in health and disease. However, the mechanisms governing exercise hyperemia that match muscle blood flow with metabolism remain poorly understood in spite of ongoing investigation since at least the 1870s. Recently, ATP has emerged as a vasodilating factor that might match O2 delivery and metabolic demand in contracting muscles. The idea is that hemoglobin in red blood cells (RBCs) releases ATP as it desaturates to cause dilation in areas of contracting muscle with high levels of O2 demand. This ATP release also opposes sympathetic vasoconstriction (functional sympatholysis) to further facilitate flow/metabolism matching. These observations, plus ATP's potent vasodilator actions, make it an attractive candidate to explain several major features of the exercise hyperemia response. In this context, we seek to understand if: a) ATP mediated vasodilation in contracting skeletal muscle is attenuated during hyperbaric hyperoxia when arterial O2 content is increased by ~25%; b) the vasodilator responses to exercise are less sensitive to changes in arterial O2 content in patients with the ?F508 mutation form of cystic fibrosis whose RBCs lack the ability to release ATP in vitro; and c) if the vasodilator responses to exercise are less sensitive to changes in arterial O2 content in the contracting muscle of healthy older volunteers who may also have altered ATP release from RBCs. In Aim 1 we will determine if ATP release is reduced during exercise with hyperbaric hyperoxia. Skeletal muscle blood flow is reduced by ~25% when arterial O2 content is increased by ~25% with hyperbaric hyperoxia. In Aim 2 we will determine if muscle blood flow is sensitive to changes in arterial O2 content in patients with CF. In Aim 3 we will determine if muscle blood flow is sensitive to changes in arterial O2 content in healthy older subjects. We will also conduct parallel in vitro studies in isolated RBCs as part of a highly mechanistic and translational experimental strategy. Our aims are designed to evaluate the relationships between forearm blood flow, O2 delivery and deep venous ATP responses during handgripping when arterial O2 content is altered by 20-25% using either hyperbaric hyperoxia or normobaric hypoxia. Our approach also leverages our prior experience with hypoxia and hyperbaric hyperoxia, ATP measurements and our history of studies in older humans and patients with CF. Thus, we are proposing innovative and novel approaches to comprehensively test the ATP hypothesis and exercise hyperemia in humans. Our studies also have the potential to identify circulating ATP, and perhaps the red blood cell, as a therapeutic target in disease states that increase with advancing age and are associated with reduced muscle perfusion (e.g. heart failure) or inadequate O2 delivery in other vascular beds. Finally, our proposal is consistent with NHLBI and NIH priorities related to translational research that seek to understand the contribution of mechanisms identified in animal models and in vitro experimental paradigms to humans.